Influenza vaccine formulation

ABSTRACT

Peptide-based anti-influenza formulations against influenza are disclosed. The peptides are derived from influenza-based epitopes. The formulations are based on peptide mixtures which may be formulated so that variability is present at particular residues. The formulations can be used to prepare vaccines for preventing influenza, particularly avian influenza.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application from U.S. patentapplication Ser. No. 11/948,505, filed Nov. 30, 2007, which claimspriority from U.S. Provisional Application Ser. No. 60/868,008 filedNov. 30, 2006, which is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates generally to an anti-viral formulation,and in particular relates to a peptide-based influenza vaccineformulation, more particularly to an avian influenza peptide-basedvaccine formulation.

BACKGROUND OF THE INVENTION

Avian influenza is an infectious disease of birds caused by type Astrains of the influenza virus. The disease, which was first identifiedin Italy more than 100 years ago, occurs worldwide. Sixteen subtypes ofinfluenza virus are known to infect birds, thus providing an extensivereservoir of influenza viruses potentially circulating in birdpopulations. To date, all known outbreaks of the highly pathogenic formhave been caused by influenza A viruses of subtypes H5 and H7.

Of the 16 avian influenza virus subtypes, H5N1 is of particular concernfor several reasons. H5N1 mutates rapidly and has a documentedpropensity to acquire genes from viruses, thereby facilitating infectionof other animal species. Indeed, its ability to cause severe disease inhumans has now been documented. Laboratory studies have demonstratedthat isolates from this virus have a high pathogenicity and can causemortality in humans.

Two other avian influenza viruses have recently been found to causeillness in humans: H7N7 and H9N2.

All type A influenza viruses are genetically labile and well adapted toelude host defences. Influenza viruses lack mechanisms for the“proofreading” and repair of errors that occur during replication. As aresult of these uncorrected errors, the genetic composition of theviruses changes as they replicate in humans and animals, and newantigenic variants emerge. These constant, permanent and usually smallchanges in the antigenic composition of influenza A viruses are known asantigenic “drift”.

Influenza viruses are typed as A or B on the basis of relatively stableintracellular nucleoproteins and envelope associated matrix proteins.Virus subtypes are based on two proteins in the viral envelope,hemagglutinin (HA) and neuraminidase (NA), which undergo constantantigenic change. 16 distinct subtypes of HA and 9 subtypes of NA arerecognized for influenza A viruses. The sudden appearance of a newsubtype (antigenic shift) has caused three major pandemics in the pastcentury: 1918 (Spanish Flu, H1N1), 1957 (Asian Flu, H2N2) and 1968 (HongKong Flu, H3N2).

Influenza viruses have a second characteristic of great public healthconcern: influenza A viruses can swap or “re-assort” genetic materialsbetween subtypes of any species resulting in novel subtypes. Thisreassortment process, known as antigenic “shift,” has resulted inworldwide pandemics in humans.

Influenza pandemics have occurred, on average, three to four times eachcentury when new virus subtypes have emerged that are readilytransmitted from person to person. In the 20th century, the greatinfluenza pandemic of 1918-1919, which caused an estimated 40 to 50million deaths worldwide, was followed by pandemics in 1957-1958 and1968-1969. Experts surmise that another influenza pandemic is inevitableand possibly imminent. Given the unpredictable behaviour of influenzaviruses, neither the timing nor the severity of the next pandemic can bepredicted with any certainty.

Seven variable B-cell epitopes, and one variable T-cell epitopecollectively represent the antigenic drift sites found on thehemagglutinin HA1 protein of Influenza A (subtype H5). Each of theB-cell variable epitopes represents a conformational epitope, and fourof them are comprised of two discontinuous stretches of amino acids.There are two extended antigenic sites on the HA1 proteins, and each ofthem is represented by two distinct peptide sequences. The nonadjacentsegments (stretches of amino acids) that are artificially joinedtogether to represent the discontinuous epitopes are selected using thethree-dimensional structure of A/duck/Singapore/3/97 hemagglutinin (PDBID code: 1JSM). Use of crystallographic data aids in design of linearpeptides that can mimic the native conformational epitopes of proteins.The T-cell eptiope is represented by a linear peptide sequence which mayalso be lipidated.

To date, no effective peptide-based vaccine against avian influenza iscommercially available.

Current antiviral therapies may be clinically effective againstinfluenza A virus strains in otherwise healthy adults and children;however, these therapies have limitations. Some of these drugs areexpensive and supplies are limited. The vaccine composition must alsochange each year to account for changes in the virus circulating in thepopulation due to antigenic drift. At least four months of developmenttime is required to produce a new effective vaccine in significantquantities.

Processes for preparation of an immunogenic peptide mixture aredescribed by Torres in U.S. Pat. No. 7,118,874, and in PCT applicationPCT/CA06/000891, herein incorporated by reference. According to one ofthese processes, the variability of immunogenic epitope sequences of apathogen are evaluated. A peptide mixture is synthesized comprising aplurality of peptides representative of the frequency with whichdifferent amino acids are found at variable residues of selectedepitopes.

Thus, there is a need to develop a vaccine formulation effective againstmultiple subtypes and multiple variants of avian influenza.

SUMMARY OF THE INVENTION

It is an object of the present invention to obviate or mitigate at leastone disadvantage of previous influenza vaccine formulations.

In a first aspect, the present invention provides a peptide-basedanti-influenza formulation comprising at least one peptide selected fromthe group consisting of SEQ ID NOs: 1 to 496 and analogues thereof.Particularly, the present invention provides a peptide-basedanti-influenza formulation comprising at least one peptide selected fromthe group consisting of SEQ ID NOs: 1 to 248 and analogues thereof. Inaddition, the present invention provides a formulation comprising atleast one peptide selected from the group consisting of SEQ ID NOs: 249to 496 and analogues thereof. In exemplary embodiments, the presentinvention provides a formulation comprising at least one peptideselected from the group consisting of SEQ ID NOs: 1 to 212, aformulation comprising at least one peptide selected from the groupconsisting of SEQ ID NOs: 249 to 460, a formulation comprising peptidesselected from the group consisting of: a) SEQ ID NOs: 1 to 212, and b)SEQ ID NOs: 249 to 460; a formulation comprising peptides selected fromthe group consisting of: a) SEQ ID NOs: 213 to 248, and b) SEQ ID NOs:461 to 496; a formulation comprising peptides selected from the groupconsisting of: a) SEQ ID NOs: 1 to 248, and b) SEQ ID NOs: 249 to 496;or a formulation comprising peptides selected from the group consistingof: a) SEQ ID NOs: 1 to 40, and b) SEQ ID NOs: 249 to 288.

In another exemplary embodiment of the present invention, theformulation comprises at least one peptide sequence from at least one ofthe following groups: a) SEQ ID NOs: 1 to 24; b) SEQ ID NOs: 25 to 40;c) SEQ ID NOs: 41 to 64; d) SEQ ID NOs: 65 to 88; e) SEQ ID NOs: 89 to120; f) SEQ ID NOs: 121 to 144; g) SEQ ID NOs: 145 to 176, h) SEQ IDNOs: 177 to 212; i) SEQ ID NOs: 249 to 272; j) SEQ ID NOs: 273 to 288;k) SEQ ID NOs: 289 to 312; l) SEQ ID NOs: 313 to 336; m) SEQ ID NOs: 337to 368; n) SEQ ID NOs: 369 to 392; o) SEQ ID NOs: 393 to 424; or p) SEQID NOs: 425 to 460.

In yet another exemplary embodiment of the present invention, theformulation comprises 2^(n) peptide sequences from at least one of thefollowing groups: a) SEQ ID NOs: 1 to 24; b) SEQ ID NOs: 25 to 40; c)SEQ ID NOs: 41 to 64; d) SEQ ID NOs: 65 to 88; e) SEQ ID NOs: 89 to 120;f) SEQ ID NOs: 121 to 144; g) SEQ ID NOs: 145 to 176, h) SEQ ID NOs: 177to 212, i) SEQ ID NOs: 249 to 272; j) SEQ ID NOs: 273 to 288; k) SEQ IDNOs: 289 to 312; l) SEQ ID NOs: 313 to 336; m) SEQ ID NOs: 337 to 368;n) SEQ ID NOs: 369 to 392; o) SEQ ID NOs: 393 to 424; or p) SEQ ID NOs:425 to 460, wherein n is 1 to 4.

The formulation can further comprise at least one peptide sequence fromSEQ ID NOs: 213 to 248, or SEQ ID NOs: 461 to 496.

In a further aspect of the present invention there is provided a vaccinecomprising the formulation comprising at least one peptide selected fromthe group consisting of SEQ ID NOs: 1 to 496 and analogues thereof,together with a pharmaceutically-acceptable diluent or carrier. Thevaccine can further comprise an adjuvant. In one example, the adjuvantis alum.

The anti-viral formulation can be an anti-influenza formulation. Moreparticularly, the anti-influenza formulation can be an aviananti-influenza formulation.

In a further aspect of the present invention, there is provided a use ofthe formulation comprising at least one peptide selected from the groupconsisting of SEQ ID NOs: 1 to 496 and analogues thereof, for thepreparation of a vaccine. The vaccine can be used for preventing ortreating influenza in an animal in need thereof. In one exemplaryembodiment, the influenza is avian influenza. The present inventionfurther relates to a method for inducing an immune response in humans oranimals and conferring protection against avian influenza, or novelsubtypes of influenza derived from avian influenza, which comprisesadministering to humans or other animals a peptide-based vaccine asdescribed herein.

In a further aspect of the present invention, there is provided a methodfor preparing an anti-viral formulation, such as the anti-viralformulation as described herein. According to one embodiment, there isprovided a method for preparing a peptide from SEQ ID NOs: 1 to 212comprising the steps of determining a linear sequence representative ofprimary sequences of discontinuous epitopes of an avian influenza viralprotein, wherein the epitopes are in proximity to each other when theprotein is in a folded conformation; and synthesizing a peptiderepresentative of the linear sequence. In another embodiment, there isprovided method for preparing a peptide mixture comprising any twopeptide sequences from SEQ ID NOs: 1 to 212 comprising the steps of:determining a linear sequence representative of primary sequences ofdiscontinuous epitopes of an avian influenza viral protein, the epitopesbeing in proximity to each other when the protein is in a foldedconformation; said discontinuous epitopes comprising variable residues,and synthesizing a peptide mixture including at least two differentamino acids at a variable residue.

In yet another aspect, the present invention relates generally to ananti-influenza vaccine comprising a mixture of peptides containing atleast one hemagglutinin (HA) antigen of influenza virus. Hemagglutinin(HA) is a potent immunogen, and viral neutralizing antibodies aredirected against the variable regions of HA. The isolated peptidemixture represents variants of multiple variable regions ofhemagglutinin. Thus, in accordance with one aspect of the presentinvention, there is provided an anti-viral formulation comprising amixture of isolated peptides, said mixture being formulated on the basisof the variable region of the avian influenza virus HA protein and saidisolated peptide mixture representing variants of a variable region ofthe HA or HA1 protein, wherein each of said variable regions comprisinga plurality of variable amino acid residues, at least one of which isrepresented by two or more amino acids.

In one embodiment, the plurality of variable amino acid residues in theanti-viral formulation comprises three or more residues. One or more ofsaid Avian influenza proteins can be an HA or HA1.

Other aspects and features of the present invention will become apparentto those ordinarily skilled in the art upon review of the followingdescription of specific embodiments of the invention in conjunction withthe accompanying Figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will now be described, by way ofexample only, with reference to the attached Figures, wherein:

FIG. 1 shows an analytical HPLC chromatogram of crude INFA-H5-1-V1peptide sequences (corresponding to SEQ ID NOs: 249 to 272).

FIG. 2 shows an analytical HPLC chromatogram of crude INFA-H5-1-V2peptide sequences (corresponding SEQ ID NOs: 273 to 288).

FIG. 3 shows an analytical HPLC chromatogram of crude INFA-H5-1-V3peptide sequences (corresponding to SEQ ID NOs: 289 to 312).

FIG. 4 shows an analytical HPLC chromatogram of crude INFA-H5-1-V4peptide sequences (corresponding to SEQ ID NOs: 313 to 336).

FIG. 5 shows an analytical HPLC chromatogram of crude INFA-H5-1-V5peptide sequences (corresponding to SEQ ID NOs: 337 to 368).

FIG. 6 shows an analytical HPLC chromatogram of crude INFA-H5-1-V6peptide sequences (corresponding to SEQ ID NOs: 369 to 392).

FIG. 7 shows an analytical HPLC chromatogram of crude INFA-H5-1-V7peptide sequences (corresponding to SEQ ID NOs: 393 to 424).

FIG. 8 shows an analytical HPLC chromatogram of crude INFA-H5-1-V8peptide sequences (corresponding to SEQ ID NOs: 425 to 460).

FIG. 9 shows a MALDI-TOF spectrum of crude INFA-H5-1-V1 peptidesequences (corresponding to SEQ ID NOs: 249 to 272).

FIG. 10 shows a MALDI-TOF spectrum of crude INFA-H5-1-V2 peptidesequences (corresponding to SEQ ID NOs: 273 to 288).

FIG. 11 shows a MALDI-TOF spectrum of crude INFA-H5-1-V3 peptidesequences (corresponding to SEQ ID NOs: 289 to 312).

FIG. 12 shows a MALDI-TOF spectrum of crude INFA-H5-1-V4 peptidesequences (corresponding to SEQ ID NOs: 313 to 336).

FIG. 13 shows a MALDI-TOF spectrum of crude INFA-H5-1-V5 peptidesequences (corresponding to SEQ ID NOs: 337 to 368).

FIG. 14 shows a MALDI-TOF spectrum of crude INFA-H5-1-V6 peptidesequences (corresponding to SEQ ID NOs: 369 to 392).

FIG. 15 shows a MALDI-TOF spectrum of crude INFA-H5-1-V7 peptidesequences (corresponding to SEQ ID NOs: 393 to 424).

FIG. 16 shows a MALDI-TOF spectrum of crude INFA-H5-1-V8 peptidesequences (corresponding to SEQ ID NOs: 425 to 460).

FIG. 17 shows a MALDI-TOF spectrum of crude lipidated INFA-H5-1-V8peptide sequences (corresponding to SEQ ID NOs: 461 to 496).

FIG. 18( a)-(h) shows the different variosite peptide sequences of thepresent invention, with variable residues beneath the consensussequence. FIG. 18( a) is INFA-H5-1-V1; FIG. 18( b) is INFA-H5-1-V2; FIG.18( c) is INFA-H5-1-V3; FIG. 18( d) is INFA-H5-1-V4; FIG. 18( e) isINFA-H5-1-V5; FIG. 18( f) is INFA-H5-1-V6; FIG. 18( g) is INFA-H5-1-V7;FIG. 18( h) is INFA-H5-1-V8.

FIG. 18( i) shows different lipidated variosite peptide sequences basedon the consensus sequence in FIG. 18( h).

FIG. 19 illustrates induction of humoral immunity by a vaccine of thepresent invention after immunization. Blue bar (top bar)=AviFlu vaccineINFA-02L+alum; Red bar (2^(nd) bar from top)=AviFlu vaccine INFA-02Lwithout adjuvant; Purple bar (middle bar)=AviFlu vaccineINFA-02P+montanide; Green bar (2^(nd) bar from bottom)=AviFlu vaccineINFA-02P+alum; Black bar (bottom bar)=control.

FIG. 20 illustrates a survival plot of vaccinated mice against challengewith H5N1. Legend from top: Black=control; Green=INFA-02P+alum;Purple=INFA-02P+montanide; Red=INFA-02L without adjuvant;Blue=INFA-02L+alum.

FIG. 21 shows induction of humoral immunity by INFA-01P(INFA-HA-1-(V1-V2)) versus INFA-02P (INFA-HA-1-(V1-V8)) aftervaccination in mice as measured by HAI titres. Blue bar (bottombar)=INFA-01P+montanide; Purple bar (middle bar)=INFA-02P+montanide;Black bar (top bar)=control.

FIG. 22 shows a survival plot of mice, vaccinated by INFA-01P(INFA-HA-1-(V1-V2)), against challenge with H5N1. Legend from top:Black=control; Blue−INFA-01P+montanide.

DETAILED DESCRIPTION

Generally, the present invention provides an anti-influenza formulation,and, more specifically, a vaccine for Influenza A, including aviansubtypes.

In a first aspect, the present invention provides a peptide-basedanti-influenza formulation comprising at least one peptide selected fromthe group consisting of SEQ ID NOs: 1 to 496 and analogues thereof.Particularly, the present invention provides a peptide-basedanti-influenza formulation comprising at least one peptide selected fromthe group consisting of SEQ ID NOs: 1 to 248 and analogues thereof. Inaddition, the present invention provides a formulation comprising atleast one peptide selected from the group consisting of SEQ ID NOs: 249to 496 and analogues thereof. In exemplary embodiments, the presentinvention provides a formulation comprising at least one peptideselected from the group consisting of SEQ ID NOs: 1 to 212, aformulation comprising at least one peptide selected from the groupconsisting of SEQ ID NOs: 249 to 460, a formulation comprising peptidesselected from the group consisting of: a) SEQ ID NOs: 1 to 212, and b)SEQ ID NOs: 249 to 460; a formulation comprising peptides selected fromthe group consisting of: a) SEQ ID NOs: 213 to 248, and b) SEQ ID NOs:461 to 496; a formulation comprising peptides selected from the groupconsisting of: a) SEQ ID NOs: 1 to 248, and b) SEQ ID NOs: 249 to 496;or a formulation comprising peptides selected from the group consistingof: a) SEQ ID NOs: 1 to 40, and b) SEQ ID NOs: 249 to 288.

In another exemplary embodiment of the present invention, theformulation comprises at least one peptide sequence from at least one ofthe following groups: a) SEQ ID NOs: 1 to 24; b) SEQ ID NOs: 25 to 40;c) SEQ ID NOs: 41 to 64; d) SEQ ID NOs: 65 to 88; e) SEQ ID NOs: 89 to120; f) SEQ ID NOs: 121 to 144; g) SEQ ID NOs: 145 to 176, h) SEQ IDNOs: 177 to 212; i) SEQ ID NOs: 249 to 272; j) SEQ ID NOs: 273 to 288;k) SEQ ID NOs: 289 to 312; l) SEQ ID NOs: 313 to 336; m) SEQ ID NOs: 337to 368; n) SEQ ID NOs: 369 to 392; o) SEQ ID NOs: 393 to 424; or p) SEQID NOs: 425 to 460.

In yet another exemplary embodiment of the present invention, theformulation comprises 2^(n) peptide sequences from at least one of thefollowing groups: a) SEQ ID NOs: 1 to 24; b) SEQ ID NOs: 25 to 40; c)SEQ ID NOs: 41 to 64; d) SEQ ID NOs: 65 to 88; e) SEQ ID NOs: 89 to 120;f) SEQ ID NOs: 121 to 144; g) SEQ ID NOs: 145 to 176, h) SEQ ID NOs: 177to 212, i) SEQ ID NOs: 249 to 272; j) SEQ ID NOs: 273 to 288; k) SEQ IDNOs: 289 to 312; l) SEQ ID NOs: 313 to 336; m) SEQ ID NOs: 337 to 368;n) SEQ ID NOs: 369 to 392; o) SEQ ID NOs: 393 to 424; or p) SEQ ID NOs:425 to 460, wherein n is 1 to 4.

The formulation can further comprise at least one peptide sequence fromSEQ ID NOs: 213 to 248 or SEQ ID NOs: 461 to 496.

In a further aspect of the present invention there is provided a vaccinecomprising the formulation comprising at least one peptide selected fromthe group consisting of SEQ ID NOs: 1 to 496 and analogues thereof,together with a pharmaceutically-acceptable diluent or carrier. Thevaccine can further comprise an adjuvant. In one example, the adjuvantis alum.

The anti-viral formulation can be an anti-influenza formulation. Moreparticularly, the anti-influenza formulation can be an aviananti-influenza formulation.

In a further aspect of the present invention, there is provided a use ofthe formulation comprising at least one peptide selected from the groupconsisting of SEQ ID NOs: 1 to 496 and analogues thereof, for thepreparation of a vaccine. The vaccine can be used for preventing ortreating influenza in an animal in need thereof. In one exemplaryembodiment, the influenza is avian influenza. The present inventionfurther relates to a method for inducing an immune response in humans oranimals and conferring protection against avian influenza, or novelsubtypes of influenza derived from avian influenza, which comprisesadministering to humans or other animals a peptide-based vaccine asdescribed herein.

In a further aspect of the present invention, there is provided a methodfor preparing an anti-viral formulation, such as the anti-viralformulation as described herein. According to one embodiment, there isprovided a method for preparing a peptide from SEQ ID NOs: 1 to 212comprising the steps of determining a linear sequence representative ofprimary sequences of discontinuous epitopes of an avian influenza viralprotein, wherein the epitopes are in proximity to each other when theprotein is in a folded conformation; and synthesizing a peptiderepresentative of the linear sequence. In another embodiment, there isprovided method for preparing a peptide mixture comprising any twopeptide sequences from SEQ ID NOs: 1 to 212 comprising the steps of:determining a linear sequence representative of primary sequences ofdiscontinuous epitopes of an avian influenza viral protein, the epitopesbeing in proximity to each other when the protein is in a foldedconformation; said discontinuous epitopes comprising variable residues,and synthesizing a peptide mixture including at least two differentamino acids at a variable residue.

In yet another aspect, the present invention relates generally to ananti-influenza vaccine comprising a mixture of peptides containing atleast one hemagglutinin (HA) antigen of influenza virus. Hemagglutinin(HA) is a potent immunogen, and viral neutralizing antibodies aredirected against the variable regions of HA. The isolated peptidemixture represents variants of multiple variable regions ofhemagglutinin. Thus, in accordance with one aspect of the presentinvention, there is provided an anti-viral formulation comprising amixture of isolated peptides, said mixture being formulated on the basisof the variable region of the avian influenza virus HA protein and saidisolated peptide mixture representing variants of a variable region ofthe HA or HA1 protein, wherein each of said variable regions comprisinga plurality of variable amino acid residues, at least one of which isrepresented by two or more amino acids.

In one embodiment, the plurality of variable amino acid residues in theanti-viral formulation comprises three or more residues. One or more ofsaid Avian influenza proteins can be an HA or HA1.

The vaccine may be formulated with or without representing variation atspecific residues for each peptide. When variation is not represented,the peptide formed may be referred to herein as a Discotope™ construct.A discotope construct is a linear sequence synthetic construct thatapproximates the position of primary sequence sections that composediscontinuous epitopes. The individual sections are constructed insequence to elicit immune responses that recognize the discontinuousepitopes found in the original intact protein.

Discontinuous epitopes are composed of two or more segments of theprimary sequence of a protein that when properly folded come togetherand are bound by specific antibodies. They are not recognized byantibodies when the secondary structure is lost and therefore have notbeen represented by a continuous linear peptide.

When variation is present at particular residues that are known to havedifferent amino acids represented according to different sequences forthat particular pathogen, the formulation comprises a number ofpeptides, which may be collectively referred to herein as a Discosite™construct.

Design of Eptiopes

Hemagglutinin is the major surface glycoprotein of influenza virus and apotent immunogen against which viral neutralizing antibodies aredirected. We have designed eight peptides that mimic discontinuous B-and T-cell epitopes on antigenic sites of HA. The sequences of thesepeptides are determined based on analysis of the crystal structure ofinfluenza hemagglutinin (HA) protein to determine peptide epitopes.Hemagglutinin is the major surface glycoprotein of influenza virus and apotent immunogen against which viral neutralizing antibodies aredirected. The linear peptide epitopes in the cocktail mimicdiscontinuous epitopes on the HA protein surface. Using bioinformaticssoftware that analyzes the antigenic variation of HA proteins fromthousands of human influenza isolates, degenerative peptide cocktailsbased on these epitopes can be prepared which represent the antigenicvariation of HA within these epitopes. Thus, the influenza vaccineformulations of the present invention comprise a cocktail of peptidesthat represent major epitopes of the HA protein.

HA is the major envelope glycoprotein of influenza virus, and mediatesthe penetration of virus into host cells. The native HA is formed by theassociation of three HA monomers which, as a precondition of virusinfectivity, are cleaved enzymatically into the amino-terminal HA1 andcarboxy-terminal HA2. Based on the three dimensional structure of HA1,antigenic sites have been mapped by determining the amino acid changesof antigenic variants. The antigenic variations were mostly seensurrounding the receptor binding region of HA. including residues aroundthe antibody inaccessible receptor binding pockets. Monoclonalantibodies to these antigenic sites neutralize influenza virusinfectivity when the exact sequences are present. Both T and B cellepitopes are found on these sites.

All amino acids changes documented in virus escape mutants, selected byMAB or other methods, were analyzed. The proteins were aligned, andposition of those amino acids was mapped onto 3-D structure ofhemagglutinin H5. The location of the epitope was roughly predicted inhemagglutinin H5 protein as the area surrounds amino acids that undergothe immune pressure. Antigenic sites were then redefined using thethree-dimensional structure of A/duck/Singapore/3/97 hemagglutinin (PDBID code: 1JSM) in a sense that antigenic determinants must be freelyaccessible for B-cell antibodies, and that different segments of sameepitope must be in close proximity to each other (for example, within 20A).

The occurrence of amino acids at variable sites within constructedepitopes was assessed by analyzing hemagglutinin HA1 strains ofInfluenza A (subtype H5) virus, available in the Los Alamos Data Base asof Jun. 28, 2005. Either 460 from all hosts or only 38 humanhemagglutinin HA1 strains were used for analysis. A variable residue wasdefined as a position in which the occurrence of the most frequent aminoacid at that position is less than 85% among all viral sequencesexamined.

A plurality of variable amino acid residues may comprise three or moreresidues, with two or more different amino acids at each variableposition.

Part or all of the peptides comprising an influenza vaccine may belipidated.

Discontinuous epitopes are composed of two or more segments of theprimary sequence of a protein that exist in close proximity when in anative, three-dimensional conformation. They are not recognized byantibodies when the secondary or tertiary structure is lost; thus,linear peptides cannot traditionally be used to represent discontinuousepitopes. Crystallographic data from influenza hemagglutinin was used todesign linear sequences that represent at least five conformationalepitopes.

From each variable epitope, the peptide length is selected, and withinthe peptide, a plurality of variable residues is selected. Each variableresidue has at least two optional amino acids, found naturally occurringin sequenced versions of the virus. In this way, a high degree ofvariability is represented. For example, three or four variable residuesmay be represented in the mixture of peptides, each having two or moredifferent amino acids represented in the sequenced database records forinfluenza variants. If two variable residues occur in a variable region,then 2² different peptides would be used in the mixture representingthat particular region. If three or four variable residues are indicatedin a hypervariable region, the number of peptides in the resultingmixture would be 2³ and 2⁴, respectively. Generally, if variable regionsconsist of A, B, C, and so on variable sites, with a, b, c, and so ondifferent amino acids at respective site, the total number of peptideswould be A^(a)×B^(b)×C^(c) and so on.

Once the proteins, variable epitopes, peptide lengths, and variableresidues are selected, the synthesis of the peptide mixtures occurs,according to any acceptable method of peptide synthesis.

Peptide mixtures are synthesized with each different peptide sequencerepresented in roughly equimolar quantities. However, there is norequirement to provide equimolar quantities of the individual peptides.

Lipidation of peptides may be conducted by any conventional oracceptable route, as would be known to those of skill in the art.Peptides need not be lipidated, but it may be advantageous for certainpeptides to be lipidated with any acceptable lipid, such as palmiticacid, so as to allow a peptide to pass through a cell membrane. Peptidesincorporating lipid may benefit from placement of a KSS motif at theC-terminal. The peptides incorporating lipid may contain 1 or more lipidmoieties, for example, two lipid moieties per peptide. Immunization withlipidated peptides may result in an enhanced cytotoxic T lymphocyte(CTL) response.

Peptides in accordance with one aspect of the present invention (i.e.,corresponding to SEQ ID NOs. 1-212 and SEQ ID NOs. 249-460) form 8groups derived from H5 antigenic sites on hemagglutinin. These groupsare identified as INFA-H5-1-V1, INFA-H5-1-V2, INFA-H5-1-V3,INFA-H5-1-V4, INFA-H5-1-V5, INFA-H5-1-V6, INFA-H5-1-V7 and INFA-H5-1-V8.The groups contain the following sequences:

Groups INFA-H5-1-V1 (SEQ ID NOs 1-24 and SEQ ID NOs. 249-272),INFA-H5-1-V3 (SEQ ID NOs 41-64 and SEQ ID NOs. 289-312), INFA-H5-1-V4(SEQ ID NOs 65-88 and SEQ ID NOs. 313-336) and INFA-H5-1-V6 (SEQ ID NOs121-144 and SEQ ID NOs. 369-392) consist of 24 peptide variants.

Groups INFA-H5-1-V5 (SEQ ID NOs 89-120 and SEQ ID NOs. 337-368) andINFA-H5-1-V7 (SEQ ID NOs 145-176 and SEQ ID NOs. 393-424) consist of 32peptide variants.

Group INFA-H5-1-V2 (SEQ ID NOs 25-40 and SEQ ID NOs. 273-288) consistsof 16 peptide variants.

Group INFA-H5-1-V8 (SEQ ID NOs 177-212 and SEQ ID NOs. 425-460) consistsof 36 peptide variants.

During typical preparation of the peptide sequences, an additionalresidue (such as a glycine residue) may be added at an end of sequence.Sequences corresponding to peptides having an additional glycine residueare shown in SEQ ID NOs: 249 to 496. The additional glycine residue hasno material effect on the function of the peptide, and the presence ofthe glycine residue is merely a product of peptide synthesis which wouldbe well understood to the person of ordinary skill in the art. Inaddition, therefore, peptides normally synthesized in this manner wouldrepresent typical examples of “analogues” (as described below) ofpeptides used in the preparation of formulations in accordance with oneaspect of the present invention.

FIGS. 1 and 9 are related to peptide group INFA-H5-1-V1. FIG. 1 shows ananalytical HPLC chromatogram of crude INFA-H5-1-V1 peptides. FIG. 9shows a MALDI-TOF spectrum of crude INFA-H5-1-V1 peptides.

FIGS. 2 and 10 are related to peptide group INFA-H5-1-V2. FIG. 2 showsan analytical HPLC chromatogram of crude INFA-H5-1-V2 peptides. FIG. 10shows a MALDI-TOF spectrum of crude INFA-H5-1-V2 peptides.

FIGS. 3 and 11 are related to peptide group INFA-H5-1-V3. FIG. 3 showsan analytical HPLC chromatogram of crude INFA-H5-1-V3 peptides. FIG. 11shows a MALDI-TOF spectrum of crude INFA-H5-1-V3 peptides.

FIGS. 4 and 12 are related to peptide group INFA-H5-1-V4. FIG. 4 showsan analytical HPLC chromatogram of crude INFA-H5-1-V4 peptides. FIG. 12shows a MALDI-TOF spectrum of crude INFA-H5-1-V4 peptides.

FIGS. 5 and 13 are related to peptide group INFA-H5-1-V5. FIG. 5 showsan analytical HPLC chromatogram of crude INFA-H5-1-V5 peptide. FIG. 13shows a MALDI-TOF spectrum of crude INFA-H5-1-V5 peptides.

FIGS. 6 and 14 are related to peptide group INFA-H5-1-V6. FIG. 6 showsan analytical HPLC chromatogram of crude INFA-H5-1-V6 peptides. FIG. 14shows a MALDI-TOF spectrum of crude INFA-H5-1-V6 peptides.

FIGS. 7 and 15 are related to peptide group INFA-H5-1-V7. FIG. 7 showsan analytical HPLC chromatogram of crude INFA-H5-1-V7 peptides. FIG. 15shows a MALDI-TOF spectrum of crude INFA-H5-1-V7 peptides.

FIGS. 8 and 16 are related to peptide group INFA-H5-1-V8. FIG. 8 showsan analytical HPLC chromatogram of crude INFA-H5-1-V8 peptide. FIG. 16shows a MALDI-TOF spectrum of crude INFA-H5-1-V8 peptides.

FIG. 17 shows a MALDI-TOF spectrum of crude lipidated INFA-H5-1-V8peptide sequences (corresponding to SEQ ID NOs: 213 to 248). SEQ ID NOs213-248 are lipidated versions of SEQ ID NOs 177-212.

FIG. 18 shows the variosites of the present invention, includingvariable amino acid residues. FIG. 18 (a) shows the peptides of groupINFA-H5-1-V1. FIG. 18 (b) shows the peptides of INFA-H5-1-V2. FIG. 18(c) shows the peptides of INFA-H5-1-V3. FIG. 18 (d) shows the peptidesof INFA-H5-1-V4. FIG. 18 (e) shows the peptides of INFA-H5-1-V5. FIG. 18(f) shows the peptides of INFA-H5-1-V6. FIG. 18 (g) shows the peptidesof INFA-H5-1-V7. FIG. 18 (h) shows the peptides of INFA-H5-1-V8.

Design of Vaccine Formulations

In the context of the present invention, a vaccine formulation is acocktail of peptides that are used in the preparation of an influenzavaccine. The vaccine can comprise the cocktail of peptides and othersubstituents known in the art that would be found acceptable forinclusion. These substituents can include, but are not limited to,adjuvants, diluents and/or carriers.

As used in the present application, a peptide “analogue” can include avariant in which one or more residues are added, deleted, inserted orsubstituted, while having no material effect on the function of thepeptide. That is, a peptide analogue in accordance with one aspect ofthe present invention should be capable of inducing an antibody orT-cell response to HA. A residue (or residues) may be added or deletedfrom either end of the peptide, deleted from within the peptide,inserted within the peptide, or substituted for one or more of theresidues within the peptide. As would be understood by a person ofordinary skill in art, one or more peptide residues may be added,deleted, inserted or substituted while still maintaining the function ofthe peptide. For example, as many as five or more residues may be addedto or removed from either end of a peptide, or inserted into a peptide,and be considered a peptide analogue within the context of the presentinvention. In a further example, a conservative substitution of one ormore residues within a peptide may result in a peptide analogue. Aswould be well understood to the skilled artisan, a conservativesubstitution includes a substitution of one amino acid residue withanother amino acid residue having one or more similar chemicalproperties, such as polarity, charge, hydrophobicity, or aromaticity,for example.

The vaccine formulations of the present invention are particularlysuitable for preparing vaccines in the treatment of avian influenza.However, it will be appreciated that any combination of peptidesequences, or formulations comprising these peptide sequences, may beused in other influenza phenotypes.

A vaccine of the present invention may be formulated from a peptidemixture with or without variation at specific residues within eachpeptide. When variation is not present, the peptide formed is referredto herein as a consensus epitope. When variation is present atparticular residues that are known to have different amino acidsrepresented according to different sequences for that particular viralvariant or subtype, the formulation comprises a number of peptides,collectively referred to as a variable epitope or “variosite”.

Peptide vaccines can be prepared with a pool of one or more peptidesequences from SEQ ID NOs: 1 to 212 or SEQ ID NOs: 249 to 460representing epitopes contained in the three-dimensional structure ofHA. The vaccines may further comprise one or more lipidated peptides,including one or more peptides from SEQ ID NOs: 213 to 248 or SEQ IDNOs: 461 to 496. The vaccines may comprise one or more discotopeconstructs (peptides containing non-variable amino acid residues) or oneor more discosite constructs (peptides containing variable amino acidresidues). A discosite construct of the present invention is derivedfrom one of these epitopes. Thus, a discosite construct formulationcomprises one or more peptide sequences derived from the epitopecontaining the variable residues.

Each discosite construct of the present invention represents 2^(x)possible peptide sequences based on x varied residues. For example, adiscosite construct having 3 or 4 variable residues represents 2³=8 or2⁴=16 sequences, respectively. Therefore, in the context of the presentinvention, a discosite construct as referred to herein includes theepitope sequence containing the variable residues and the one or morepossible sequences derived therefrom. It will be appreciated by theperson of ordinary skill in the art that additional sequences may or maynot be added as required.

The vaccine may be prepared by any methodology acceptable to one skilledin the art. For example, oligonucleotides encoding these peptides may beinserted in viral or non-viral vectors for delivery. Peptides may besynthesized individually in and mixed together to accomplish anacceptable formulation. Any variety of different modes by which thesepeptide antigens may be prepared is acceptable for use with theinvention.

In order to formulate a vaccine that is subtype-specific, the proteinsselected may contain variable regions with selected variable amino acidsthat are characteristic of the variability found within the subtype ofinterest. This allows the vaccine to be subtype specific, which may havethe advantage of better representing the antigenic variation amongvariants within said subtype. To formulate a vaccine that has lesssubtype distinctiveness, the final peptide formulation may comprise thedifferent subtype specific formulations. For example, a vaccineformulated against avian flu could target variable residues particularto subtype H5 sequences. A vaccine formulated against human flu couldtarget variable residues characteristic of subtypes 1, 2 and/or 3sequences.

In order to formulate a vaccine that is species-specific, the proteinsselected may contain variable regions with selected variable amino acidsthat are characteristic of the variability found within the species ofinterest. This allows the vaccine to be species specific, which may havethe advantage of better representing the antigenic variation amongvariants within said species. For example, a vaccine formulated againstavian flu could target variable residues particular to avian H5sequences. A vaccine formulated against human flu could target variableresidues characteristic of both, human and avian H5 sequences.

As a specific example, the anti-INF vaccine may include the followingisolated peptides: SEQ ID NOs: 1 to 40, SEQ ID NOs: 249 to 288, orpeptide analogues thereof, in combination with a pharmaceuticallyacceptable carrier.

An exemplary anti-INF formulation may comprise one or more of, or all ofSEQ ID NOs: 1 to 212, SEQ ID NOs. 249 to 460 or peptide analoguesthereof; in combination with a pharmaceutically acceptable carrier. Theformulation may also comprise one or more lipidated peptides of SEQ IDNOs: 1 to 212 or SEQ ID NOs: 249 to 460, such as, for example, one ormore of SEQ ID NOs: 213 to 248 or SEQ ID NOs: 461 to 496.

Although all peptides of SEQ ID NOs: 1 to 212 or SEQ ID NOs: 249 to 460may be used in combination as the vaccine formulation, sub-groups ofthese peptides could be used together according to the invention. Forexample, a formulation may comprise at least one peptide sequence fromat least one of the following groups: a) SEQ ID NOs: 1 to 24; b) SEQ IDNOs: 25 to 40; c) SEQ ID NOs: 41 to 64; d) SEQ ID NOs: 65 to 88; e) SEQID NOs: 89 to 120; f) SEQ ID NOs: 121 to 144; g) SEQ ID NOs: 145 to 176,h) SEQ ID NOs: 177 to 212, i) SEQ ID NOs: 249 to 272; j) SEQ ID NOs: 273to 288; k) SEQ ID NOs: 289 to 312; l) SEQ ID NOs: 313 to 336; m) SEQ IDNOs: 337 to 368; n) SEQ ID NOs: 369 to 392; o) SEQ ID NOs: 393 to 424;or p) SEQ ID NOs: 425 to 460. Further, a vaccine of the presentinvention may comprise a formulation comprising 2^(n) peptide sequencesfrom at least one of the following groups: a) SEQ ID NOs: 1 to 24; b)SEQ ID NOs: 25 to 40; c) SEQ ID NOs: 41 to 64; d) SEQ ID NOs: 65 to 88;e) SEQ ID NOs: 89 to 120; f) SEQ ID NOs: 121 to 144; g) SEQ ID NOs: 145to 176, h) SEQ ID NOs: 177 to 212, i) SEQ ID NOs: 249 to 272; j) SEQ IDNOs: 273 to 288; k) SEQ ID NOs: 289 to 312; l) SEQ ID NOs: 313 to 336;m) SEQ ID NOs: 337 to 368; n) SEQ ID NOs: 369 to 392; o) SEQ ID NOs: 393to 424; or p) SEQ ID NOs: 425 to 460, wherein n is 1 to 4.

EXAMPLES Peptide Synthesis

The peptides were synthesized by solid phase peptide synthesis (SPPS)using 9-fluoroenylmethoxycarbonyl (Fmoc) chemistry on Pioneer™ automatedpeptide synthesizer, utilizing pre-loaded Fmoc protected NovaSyn™ TGTresin (NovaBiochem) as described. Where variability at a given positionis desired, mixture of two amino acids is placed at that position. Thisis repeated each time during the synthesis wherever the variability isdesired. While 1M solution of 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) andN-Hydroxybenzotriazole (HOBt) in dimethylformamide (DMF), and 1 Msolution of diisopropylethyl amine (DIPEA) in OMF was used for couplingamino acids, 20% piperidine in DMF was used for deblocking amino acidsduring the synthesis. Coupling was allowed to occur for one hour at roomtemperature. After the last amino acid was coupled, the resin was takenout from synthesizer and washed on a sintered glass funnel several timeswith OMF, with 2-propanol and with dichloromethylene (DCM), and driedunder high vacuum. The peptide mixtures are cleaved and deprotected bythe addition of a solution containingTFA/water/phenol/thioanisole/EDT/TIS [82:5:5:5:2:1]. The resin wasincubated at room temperature for 4 hours. Cleavage mixture was thenfiltered under reduced pressure into a flask containing a 10-fold volumeof cold ether. Resin was also rinsed twice with TFA into the same ethersolution. Following incubation for 30 minutes in a freezer to furtherassist precipitation, the sample was centrifuged at 1,000×g for 5minutes, and the ether removed. This extraction process was repeatedthree times. Following a final ether extraction, the residual organicsolvent was evaporated under nitrogen gas, and the peptide mixture wasredissolved in water and purified by using high performance liquidchromatography (HPLC). Excess of the solvent was removed by using arotor evaporator, and finally lyophilized to dry powder. Massspectrometry and amino acid analysis were performed on all theDiscotopes to ensure that they have the appropriate peptide content.

Lipidation is performed as follows. Upon completion of the synthesis ofa mixed peptide formulation on the synthesiser, the resin is removedfrom the column and placed into a vial. Dissolve 10 eq. of the PalmiticAcid, 10 eq. of TBTU and 10 eq. HOBT (all relative to the resin) in DMF(10 ml/0.1 mmol resin). Add the solution to the peptidyl resin in thevial. Add 20 eq. (relative to the resin) of the DIPEA. Adjust pH to 8-9by adding DIPEA drop-wise. Seal the vial with a screw cap and shake themixture overnight (at least 12 hours).

Vaccine Efficacy

Vaccine formulations comprising the peptide sequences of the presentinvention were tested in mice. The vaccines used are as follows:

INF-01P consists of two variosites INFA-H5-1-V1 and INFA-H5-1-V2 (SEQ IDNOs: 249-288), also referred as AviFlu-2(Montanide).

INF-02P consists of 8 variosites INFA-H5-1-V1 to INFA-H5-1-V8 (SEQ IDNOs: 249-460), also referred as AviFlu-8(Montanide), AviFlu(Montanide),AviFlu(Alum).

INF-02L consist of 9 variosites INFA-H5-1-V1 to INFA-H5-1-V8L (SEQ IDNOs: 249-496), also referred as AviFlu(Lipidated),AviFlu(Lipidated/Alum).

FIG. 19 illustrates induction of humoral immunity by a vaccine of thepresent invention after immunization.

FIG. 20 illustrates a survival plot of mice vaccinated with a vaccine ofthe present invention against challenge with H5N1.

FIG. 21 shows induction of humoral immunity by INFA-01P(INFA-HA-1-(V1-V2)) versus INFA-02P (INFA-HA-1-(V1-V8)) aftervaccination in mice as measured by HAI titres.

FIG. 22 shows a survival plot of mice, vaccinated by INFA-01P(INFA-HA-1-(V1-V2)), against challenge with H5N1.

The above-described embodiments of the present invention are intended tobe examples only. Alterations, modifications and variations may beeffected to the particular embodiments by those of skill in the artwithout departing from the scope of the invention, which is definedsolely by the claims appended hereto.

REFERENCES

-   1. Philpott, M. et al., Journal of virology (1990), 64(6),    2941-2947.-   2. Kaverin, N. et al., Journal of General Virology (2002), 83,    2497-2505.-   3. Hioe, C. et al., Journal of Virology (1990), 64(12), 6246-6251.-   4. Ha, Y. et al., Proceedings of the National Academy of Sciences,    USA (2001), 98, 11181-11186.-   5. Macken, C. et al., “The value of a database in surveillance and    vaccine selection.” in Options for the Control of Influenza    IV. A. D. M. E. Osterhaus, N. Cox & A. W. Hampson (Eds.) Amsterdam:    Elsevier Science, 2001, 103-106.

1. A peptide-based immunogenic composition comprising a peptide which isSEQ ID NO:
 25. 2. The composition of claim 1 further comprising at leastone peptide selected from the group consisting of SEQ ID NOs: 26 to 40.3. The composition of claim 1 further comprising apharmaceutically-acceptable diluent or carrier.
 4. The composition ofclaim 3 further comprising an adjuvant.
 5. The composition of claim 4wherein the adjuvant is alum.
 6. An immunogenic composition comprising amixture of isolated peptides selected from the group consisting of SEQID NOs: 25 to 40, said peptides representing variants of at least onevariable region of an avian influenza virus HA or HA1 protein, whereineach of said variable regions comprises one or more variable amino acidresidues, at least one of said variable amino acid residues isrepresented by two or more amino acids.
 7. The immunogenic compositionof claim 6, wherein said one or more variable amino acid residues isrepresented by three or more amino acids.
 8. The immunogenic compositionof claim 6, further comprising a peptide selected from the groupconsisting of SEQ ID NOs: 1 to 24 and 41 to
 212. 9. The composition ofclaim 1, further comprising a peptide selected from the group consistingof SEQ ID NOs: 1 to 24 and 41 to
 212. 10. The composition of claim 1,comprising peptides of SEQ ID NOs: 25 to
 40. 11. The composition ofclaim 1, comprising peptides of SEQ ID NOs: 1 to
 212. 12. A method ofpreventing or treating influenza in a patient, comprising administeringto the patient an immunogenically effective amount of the immunogeniccomposition according to claim
 1. 13. A method of preventing or treatinginfluenza in a patient, comprising administering to the patient animmunogenically effective amount of the composition according to claim6.
 14. A kit for use in the prevention or treatment of influenza in apatient, comprising the immunogenic composition according to claim 1 andinstructions for use of the immunogenic composition according to claim 1in the prevention or treatment of influenza.
 15. A kit for use in theprevention or treatment of influenza in a patient, comprising thecomposition according to claim 6 and instructions for use of thecomposition according to claim 6 in the prevention or treatment ofinfluenza.